PROJECT SUMMARY Humans and well-established research organisms lack the ability to regenerate their reproductive cells (germ cells) and reproductive organs. Research findings from these organisms established the current view that germ cells are a distinct lineage separated from the soma; therefore, the loss of germ cells renders an organism infertile because new germ cells cannot be derived from the soma. Contradicting this widely accepted view is the fact that many organisms (e.g. hydra, flatworms, segmented worms, and sea stars) can readily regenerate germ cells. However, the cellular source of regenerated germ cells in these organisms is very poorly understood. The goal of my laboratory is to close this knowledge gap and define the cellular origins and molecular mechanisms of germ cell regeneration. Addressing this is not feasible using established research organisms like mice, fruit flies, and nematodes, because they do not regenerate germ cells. Furthermore, many of the organisms that can regenerate germ cells are not conducive to studying the mechanisms of this process because they lack transgenic tools, or their anatomies present technical challenges such as large and opaque bodies, or inaccessibility of reproductive organs. These limitations prevent the use of live imaging ? a key tool to trace the lineages of germ cells and their source cells. We use a segmented worm, Platynereis dumerilii, for studying germ cell regeneration. Platynereis is well-suited for this study because germ cell regeneration can be induced and is achieved quickly; transcriptome databases, a draft genome, and transgenic tools (critical for genetic lineage tracing) are available; and a small and transparent body makes it excellent for live imaging. Therefore, Platynereis is a research organism that presents a rare opportunity to combine the modern techniques required to study germ cell regeneration (live-imaging, genetic lineage tracing, transcriptomics) in the relevant post-embryonic life stages (i.e. juveniles, adults) which are typically challenging to image live. We postulate three possible models for the cellular sources for the regenerated germ cells: 1) pluri/multipotent stem cells regenerate both somatic and germ cells; 2) a lineage-restricted cell population is dedicated to regenerating only the germ cells; or 3) somatic cells transdifferentiate into germ cells by reprogramming. Our experimental approach will be to: a) Test between the three cellular models of germ cell regeneration via genetic cell lineage tracing and live imaging. This will allow us to identify the exact cell lineages that give rise to germ cells during development and regeneration. b) Identify the molecular changes taking place during reprogramming source cells into germ cells by single cell RNA sequencing. This will allow us to obtain transcriptome trajectories over the course of regeneration and identify cell type-specific markers in the source cells, the intermediate states, and the new germ cells. These markers will be tested for function in future studies. The project will significantly contribute to our fundamental understanding of germ cell biology and the soma-germ cell distinction.